Formate Hydrogenlyase Pathway Research Articles

Transcriptional Regulation of Genes Involved in 3-Hydroxypropionic Acid Production in Response to Aeration of Recombinant Klebsiella pneumoniae.

We have described a metabolically engineered strain of Klebsiella pneumoniae/pUC18kan-aldHec that could convert glycerol to 3-hydroxypropionic acid (3-HP) effectively. In this study, the transcription of the genes encoding aldehyde dehydrogenase (AldH) and glycerol dehydratase (DhaB) responsible to 3-HP-biosynthesis, key enzymes in PP pathway and TCA cycle, as well as hydrogenases was observed under anaerobic, microaerobic, and aerobic conditions using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). During the fast growth and slow growth phases of cell growth, the aldH transcription levels were 7.0- and 2.1-fold enhanced under microaerobic condition, and 12.4- and 0.14-fold under fully aerobic condition, compared to the anaerobic control, while transcription of dhaB showed a downregulated manner due to aeration. Moreover, aeration increased the transcription levels of zwf, sucA, and sdhAB during the fast growth phase of the cell growth. The results also indicated that the hydrogenase 3 (hycBCDFG) and formate dehydrogenase (fdhA) mediated formate hydrogen lyase pathway was the main route to produce hydrogen and consume the excess NADH. These characteristics are useful for understanding and improving 3-HP biosynthesis in K. pneumoniae.

The role of the ferric-uptake regulator Fur and iron homeostasis in controlling levels of the [NiFe]-hydrogenases in Escherichia coli

Escherichia coli when growing anaerobically synthesizes three [NiFe]-hydrogenases. We investigated the consequences on hydrogenase levels, enzyme activity and gene expression of deleting the ferric iron- uptake regulator Fur, which is required to coordinate intracellular iron levels. Total hydrogenase activity was reduced between 80 and 90% in the fur mutant. Hydrogen production by the formate hydrogenlyase pathway was strongly reduced. Analysis of lacZ fusions to the various hydrogenase structural operons demonstrated that regulation of hya and hyb was not transcriptional, while the effect of the fur mutation on hyc was partly due to reduced intracellular formate. Immunological analysis of the hydrogenase large subunits revealed that the absolute levels of the enzymes were reduced suggesting that either post-transcriptional or post-translational control, possibly through enhanced enzyme turnover, was a major cause of reduced activity. A mutant defective in multiple iron transport systems also essentially lacked hydrogenase activity highlighting the importance of intracellular iron availability in regulating hydrogenase synthesis.

Suppression of Escherichia coli formate hydrogenlyase activity by trimethylamine N-oxide is due to drainage of the inducer formate.

The effect of the addition of trimethylamine N-oxide (TMAO) in the growth medium on Escherichia coli anaerobic fermentative and respiratory pathways was examined. Formate dehydrogenase H (FDH-H) activity was totally repressed by the addition of 40 mM TMAO, whereas the overall hydrogenase (HYD) activity was reduced by 25%. Accordingly, expression of lacZ operon fusions with the fdhF and hycB structural genes specifying FDH-H and HYD3 was reduced sevenfold and eightfold, respectively, leading to suppression of an active formate hydrogenlyase system. In contrast, global respiratory formate-dependent phenazine methosulphate reductase (FDH-PMS) activity, which consists of both the major anaerobic FDH-N enzyme and the aerobic FDH-Z isoenzyme, was increased approximately twofold. This was corroborated by a 2.5-fold stimulation of the sole fdoG-uidA transcriptional fusion which reflects the synthesis of the respiratory aerobic FDH-Z enzyme. In fdhD, fdhE or torA mutants lacking either FDH-PMS activity or TMAO reductase (TOR) activity, the formate hydrogenlyase pathway was no longer inhibited by TMAO. In addition, introduction of 30 mM formate in the growth medium was found to relieve the repressive effect of TMAO in the wild-type strain. When TMAO was added as terminal electron acceptor a significant enhancement of anaerobic growth was observed with the wild-type strain and the fdoG mutant. It was associated with the concomitant suppression of the formate hydrogenlyase enzymes. This was in contrast to the fdnG and torA mutants whose growth pattern and fermentative enzymes remained unaffected. Taken together, these results strongly suggest that formate-dependent reduction of TMAO via FDH-N and TOR reduces the amount of formate available for induction of the formate hydrogenlyase pathway.

The hydrogenases and formate dehydrogenases ofEscherichia coli

Escherichia coli has the capacity to synthesise three distinct formate dehydrogenase isoenzymes and three hydrogenase isoenzymes. All six are multisubunit, membrane-associated proteins that are functional in the anaerobic metabolism of the organism. One of the formate dehydrogenase isoenzymes is also synthesised in aerobic cells. Two of the formate dehydrogenase enzymes and two hydrogenases have a respiratory function while the formate dehydrogenase and hydrogenase associated with the formate hydrogenlyase pathway are not involved in energy conservation. The three formate dehydrogenases are molybdo-selenoproteins while the three hydrogenases are nickel enzymes; all six enzymes have an abundance of iron-sulfur clusters. These metal requirements alone invoke the necessity for a profusion of ancillary enzymes which are involved in the preparation and incorporation of these cofactors. The characterisation of a large number of pleiotropic mutants unable to synthesise either functionally active formate dehydrogenases or hydrogenases has led to the identification of a number of these enzymes. However, it is apparent that there are many more accessory proteins involved in the biosynthesis of these isoenzymes than originally anticipated. The biochemical function of the vast majority of these enzymes is not understood. Nevertheless, through the construction and study of defined mutants, together with sequence comparisons with homologous proteins from other organisms, it has been possible at least to categorise them with regard to a general requirement for the biosynthesis of all three isoenzymes or whether they have a specific function in the assembly of a particular enzyme. The identification of the structural genes encoding the formate dehydrogenase and hydrogenase isoenzymes has enabled a detailed dissection of how their expression is coordinated to the metabolic requirement for their products. Slowly, a picture is emerging of the extremely complex and involved path of events leading to the regulated synthesis, processing and assembly of catalytically active formate dehydrogenase and hydrogenase isoenzymes. This article aims to review the current state of knowledge regarding the biochemistry, genetics, molecular biology and physiology of these enzymes.

Mutational analysis and characterization of the Escherichia coli hya operon, which encodes [NiFe] hydrogenase 1

Deletion mutants of Escherichia coli specific for hydrogenase isoenzyme 1 (HYD1) have been constructed and characterized. The hya operon, which contains genes for the two HYD1 structural subunits and four additional genes, was mapped at 22 min on the E. coli chromosome. The total hydrogenase activities of the HYD1-negative mutant and wild-type strains were similar. However, the formate dehydrogenase activity associated with the formate hydrogen lyase pathway was lower in the mutant. The hya mutant (strain AP1), complemented with only the hydrogenase structural genes (hyaAB), produced antigenically identifiable but inactive HYD1 protein. The first five genes of hya (hyaA to hyaE) were required for the synthesis of active HYD1, but wild-type levels of HYD1 activity were restored only when mutant cells were transformed with all six genes of the operon. When AP1 was complemented with hya carried on a high-copy-number plasmid, the HYD1 structural subunits were overexpressed, but the excess protein was unprocessed and localized in the soluble fraction of the cell. The products of hyaDEF are postulated to be involved in the processing of nascent structural subunits (HYAA and HYAB). This processing takes place only after the subunits are inserted into the cell membrane. It is concluded that the biosynthesis of active HYD1 is a complex biochemical process involving the cellular localization and processing of nascent structural subunits, which are in turn dependent on the insertion of nickel into the nascent HYD1 large subunit.

Complementation of Escherichia coli sigma 54 (NtrA)-dependent formate hydrogenlyase activity by a cloned Thiobacillus ferrooxidans ntrA gene

The ntrA gene of Thiobacillus ferrooxidans was cloned by complementation of an Escherichia coli ntrA mutant that was unable to produce gas via the sigma 54 (NtrA)-dependent formate hydrogenlyase pathway. Analysis of the DNA sequence showed that the T. ferrooxidans ntrA gene coded for a protein of 475 amino acids (calculated Mr, 52,972). The T. ferrooxidans NtrA protein had 49, 44, 33, and 18% amino acid similarity with the NtrA proteins of Klebsiella pneumoniae, Azotobacter vinelandii, Rhizobium meliloti, and Rhodobacter capsulatus, respectively. The ability of the T. ferrooxidans NtrA protein to direct transcription from sigma 54-dependent promoters was demonstrated in E. coli by using fdhF-lacZ and nifH-lacZ fusions. An open reading frame coding for a protein of 241 amino acids (calculated Mr, 27,023) was situated 12 base pairs upstream of the T. ferrooxidans ntrA gene. Comparison of this protein with the product of the open reading frame ORF1, located upstream of the R. meliloti ntrA gene, showed that the two proteins had 55% amino acid similarity. The cloned T. ferrooxidans ntrA gene was expressed in E. coli from a promoter located within the ORF1 coding region.

Partial characterization of an electrophoretically labile hydrogenase activity of Escherichia coli K-12.

A mutant of Escherichia coli K-12 is described that is specifically impaired in only one hydrogenase isoenzyme. By means of Tn5-mediated insertional mutagenesis, a class of mutants was isolated (class I) that had retained 20% of the overall hydrogenase activity. As determined by neutral polyacrylamide gel electrophoresis, the mutant contained normal amounts of the hydrogenase isoenzymes 1 and 2. Therefore, the hydrogenase activity affected seemed to be electrophoretically labile and was called hydrogenase L. The presence of such an activity was recently suggested in various papers and was called isoenzyme 3. Hydrogenase L might be identical or part of the latter isoenzyme. By DEAE ion-exchange chromatography it could be separated from hydrogenases 1 and 2. Hydrogenase activity in the parent strain HB101, determined manometrically with cell-free preparations and methylviologen as the electron acceptor, immediately showed maximal activity. However, class I mutants showed a lag phase which was dependent on the protein concentration utilized in the assay. This suggested that the fast initial activity of HB101 was due to hydrogenase L. The enzyme or enzyme complex showed an Mr around 300,000 and a pH optimum between 7 and 8. Strong indications about its physiological role were provided by the finding that in class I mutants H2 production by the formate-hydrogen lyase pathway was unimpaired, whereas fumarate-dependent H2 uptake was essentially zero. Complementation with F-prime factor F'116 but not with F'143 and coconjugation and cotransduction experiments localized the mutation (hydL) close to metC at approximately 64.8 min.

Regulation of the fdhF gene encoding the selenopolypeptide for benzyl viologen-linked formate dehydrogenase in Escherichia coli.

Two classes of mutants defective in benzyl viologen-linked formate dehydrogenase (FDH-BV) activity were isolated from Escherichia coli K12. Class I consisted of four mutants which were specifically devoid of FDH-BV activity. Their mutation mapped between the ssb and melA genes at 92 min on the genome, at a site recently designated fdhF by Pecher et al. (1985). The direction of transcription of gene fdhF was found to be counterclockwise on the E. coli chromosome in one Mudl(Aprlac) fusion mutant. Expression of the lac operon in this mutant was induced by formate and repressed by nitrate, nitrite or trimethylamine N-oxide. It was found to be dependent on the positive control exerted by the fdhA, B and C genes, possibly involved in selenium incorporation, and by an hydB gene affecting the formate hydrogenlyase pathway. Class II, represented by one Mudl(Aprlac) mutant, exhibited no FDH-BV activity and a reduced level of hydrogenase activity. The relevant fdv mutation was shown to be located at 58 min and to affect the expression of fdhF.

Resolution of distinct selenium-containing formate dehydrogenases from Escherichia coli

Formate dehydrogenase, a component activity of two alternative electron transport pathways in anaerobic Escherichia coli, has been resolved as two distinguishable enzymes. One, which was induced with nitrate reductase as a component of the formate-nitrate reductase pathway, utilized phenazine methosulfate (PMS) in preference to benzyl viologen (BV) as an artificial electron acceptor and appeared to be exclusively membrane-bound. A second formate dehydrogenase, which was induced as a component of the formate hydrogenlyase pathway, appeared to exist both as a membrane-bound form and as a cytoplasmic enzyme; the cytoplasmic activity was resolved completely from the PMS-linked activity on a sucrose gradient. When E. coli was grown in the presence of 75Se-selenite, a 110,000-dalton selenopeptide, previously shown to be a component of the PMS-linked enzyme, was induced and repressed with this activity. In contrast, an 80,000-dalton selenopeptide was induced and repressed with the BV-linked activity and exhibited a distribution similar to the BV-linked formate dehydrogenase in cell fractions and in sucrose gradients. The results indicate that the two formate dehydrogenases are distinguishable on the basis of their artificial electron acceptor specificity, their cellular localization, and the size of their respective selenoprotein components.

Immunochemical analysis of the membrane-bound hydrogenase of Escherichia coli

The membrane-bound hydrogenase [EC 1.12.-.-l ofEscherichia coli is involved in the energy-conserving oxidation of hydrogen [l-4] via fumarate reductase [EC 1.3.99.11 and also in the formate hydrogenlyase pathway which converts formate to COZ and Hz [5]. E. coli hydrogenase from aerobically grown cells has been isolated and characterised [6] and the enzyme from anaerobically grown cells has been partially characterised [7]. Antibodies specific for Bacillus subtilis membrane-bound succinate dehydrogenase [EC 1.3.99.11 have been raised from activity-stained precipitin arcs located after analysis of crude fractions by crossed immunoelectrophoresis using antisera raised to detergent-solubilised membranes [8]. We have used a similar approach to prepare antibodies specific for E. coli hydrogenase and its use has enabled the subunit MW of the enzyme from anaerobically grown cells to be determined. We also report the isolation and immunological characterisation of two new E. coli mutants which specifically lack hydrogenase activity.